Electron transfer reactions play a key role in oxidative phosphorylation and respiration, drug and carcinogen metabolism and activation, immune response, collagen synthesis, and intermediary metabolism. Our aim is to understand in detailed mechanistic terms the electron transfer reactions of multisite metalloenzymes that catalyze the four-electron reduction of molecular oxygen to water. The enzymes we have chosen for investigation are blue copper oxidases (the laccases) that contain four coppers (per mol) distributed in three spectroscopically distinct sites. To lay a foundation for this work, we also intend to examine the electron transfer mechanisms employed by plastocyanin, which is a structurally well-characterized, single-site copper protein. Our long-term objective is to determine the factors that control the thermodynamics and kinetics of the electron transfer reactions of the laccases. During the next three years we propose to: (1) determine by emission lifetime and laser flash experiments thte rates of electron transfer from copper(I) in plastocyanin to electronically excited polypyridyl ruthenium(II) and chromium(III) reagents attached several angstroms away at surface binding sites (the back electron transfer rates, e.g., RU(I)greater than Cu(II), will also be measured), thereby providing important information relating directly to electron tunneling through proteins; (2) measure the temperature dependences of the reduction potentials of tree and fungal laccases by spectroelectrochemical techniques; and (3) perform chemical modification and 500 MHz proton NMR experiments aimed at elucidating the structural nature of the histidines in tree and fungal laccases.